Bis-transition-metal-chelate probes

ABSTRACT

A molecule for labeling a target material is provided including two transition-metal chelates and a detectable group. The molecule has the general structural formula (I):  
                 
         wherein: (a) Y and Y′ are each a transition metal, (b) R 1  and R 1′  are each independently CH(COO − ), CH(COOH), or absent; (c) R 2  and R 2′  are linkers each having a length of from about 3.0 to about 20 Å; and (d) X is a detectable group. The linkers may be linear or branched, may contain aromatic moieties, and may optionally be further substituted. Methods of using the molecules of the invention as probes in detecting and analyzing target materials as well as kits including the molecule of the invention are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.10/665,227, filed Sep. 17, 2003, which is a continuation-in-part ofInternational Application No. PCT/US02/36180 filed on Nov. 12, 2002,which claims the benefit of U.S Provisional Application No. 60/410,267,filed Sep. 13, 2002 and U.S. Provisional Application No. 60/367,775,filed Mar. 28, 2002, the contents all of which are herein incorporatedby reference.

This invention was made with Government support under Grant No. NIHR01-GM41376, awarded by the National Institutes of Health. Therefore,the Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to inventive molecules and methods useful inlabeling target molecules. More particularly, the present inventionrelates to certain transition metal chelate molecules capable ofselectively associating with histidine-containing target sequences ontarget materials or compounds of interest and yielding a detectablesignal. The invention further relates to kits for use of the inventivetransition metal chelate molecules.

BACKGROUND OF THE INVENTION

Characterization of proteins often requires the ability to incorporatedetectable groups—e.g., fluorochromes, chromophores, spin labels,radioisotopes, paramagnetic atoms, heavy atoms, haptens, crosslinkingagents, and cleavage agents—at specific, defined sites. For proteinsthat do not contain pre-existing cysteine residues, site-specificlabeling can be accomplished by use of site-directed mutagenesis tointroduce a cysteine residue at the site of interest, followed bycysteine-specific chemical modification to incorporate the labeledprobe. However, for proteins that contain pre-existing cysteineresidues, site-specific labeling is difficult. Multiple strategies havebeen reported: (i) intein-mediated labeling (“expressed proteinligation”), (Muir, et al., Proc. Nat'l. Acad. Sci. USA, 95:6705-6710(1998)); (ii) transglutaminase-mediated labeling (Sato et al., Biochem.35:13072-13080 (1996)); (iii) oxidation-mediated labeling (Geoghegan, etal., Bioconj. Chem., 3:138-146 (1992)); and (iv)trivalent-arsenic-mediated labeling (Griffin et al., Science281:269-272, 1998) (U.S. Pat. No. 6,008,378). Strategies (i)-(iii) donot permit in situ labeling (i.e., direct labeling of proteins incuvettes, gels, blots, or biological samples—without the need for asubsequent purification step) or in vivo labeling (i.e., direct labelingof proteins in cells). Strategy (iv) requires a structural scaffoldpresenting two trivalent-arsenic atoms in a precisely defined spatialrelationship and therefore relates only to a limited number ofdetectable groups (such as those having a detectable xanthene,xanthanone, or phenoxazinestructural nucleus).

Transition-metal chelates consisting of a transition-metal ion, such asNi²⁺, Co²⁺, Cu²⁺, or Zn²⁺, in complex with a tridentate or tetradentatechelating ligand, such as iminodiacetic acid (IDA) or nitrilotriaceticacid (NTA), exhibit high affinity for oligohistidine sequences,particularly hexahistidine sequences (Sulkowski, E., Trends Biotechnol.,3:1-7 (1985); Hochuli, et al., J. Chromat. 411:177-184 (1987); Hochuli,E. et al. BioTechnol. 6:1321-1325 (1988). FIG. 1 shows a proposed modelfor binding of a neighboring hexahistidine residue to a Ni-NTA resin asdisclosed in Crowe, J. et al., Methods Mol. Biol., 31:371-387 (1994)).

The high affinity of interactions between transition-metal chelates andoligohistidine sequences, particularly hexahistidine sequences, has beenverified using force microscopy experiments, which permit directmeasurement of interaction forces on the single-molecule level anddirect observation of molecular recognition of a single receptor-ligandpair (Kienberger, F. et al. Single Mol. 1:59-65 (2000); Schmitt, L. etal. Biophys. J. 78: 3275-3285 (2000)).

The high affinity of interactions between transition-metal chelates andoligohistidine sequences, particularly hexahistidine sequences, has beenused advantageously to purify biomolecules containing, or modified tocontain, “oligohistidine tags,” particularly “hexahistidine tags”(Hochuli, E. et al. BioTechnol. 6:1321-1325 (1988); Crowe, J. et al.,Methods Mol. Biol., 31:371-387 (1994)). In this application, termed“immobilized-metal-chelate affinity chromatography,” a transition-metalchelate consisting of a transition-metal ion, such as Ni²⁺, Co²⁺,Cu^({overscore (2)}+), or Zn²⁺, in complex with a tridentate ortetradentate chelating ligand, such as iminodiacetic acid (IDA) ornitrilotriacetic acid (NTA), is immobilized on a solid phase, such aschromatographic resin, and the resulting immobilized metal chelate isused to bind, and thereby purify from other components, taggedbiomolecules.

The high affinity of interactions between transition-metal chelates andoligohistidine tags, particularly hexahistidine tags, also has been usedadvantageously in biosensor analysis of biomolecules containing, ormodified to contain, oligohistidine tags, particularly hexahistidinetags (Gershon, et al. J. Immunol. Meths. 183:65-76 (1995); Nieba, L. etal. Anal. Biochem. 252:217-228 (1997)). Kienberger et al., Single Mol.1; S9-65 (2000). In this application, a transition-metal chelateconsisting of a transition-metal ion, such as Ni²⁺, Co²⁺, Cu²⁺, or Zn²⁺,in complex with a tridentate or tetradentate chelating ligand, such asiminodiacetic acid (IDA) or nitrilotriacetic acid (NTA), is immobilizedon a biosensor chip, such a surface-plasmon-resonance biosensor chip,and the resulting immobilized metal chelate is used to detect, quantify,and analyze tagged biomolecules.

It would be advantageous to be able to use the high affinity ofinteractions between transition-metal chelates and oligohistidine tags,particularly hexahistidine tags, in labeling and in situ detection oftagged target materials, in particular, biomolecules.

There is a need for improved methods and compositions for proteinlabeling. In particular, there is a need for methods and compositionsthat permit in situ labeling, that permit in vivo labeling, and thatencompass a wide range of detectable groups with different properties.

SUMMARY OF THE INVENTION

The invention provides a molecule with two pendant metal-chelatemoieties according to the general structural Formula (I), includingtautomers, salts, and acids thereof:

wherein: (a) Y and Y′ are each a transition metal, (b) R¹ and R^(1′) areeach independently C(COO⁻), CH(COOH), or absent; (c) R² and R^(2′) arelinkers each having a length of from about 3.0 to about 20 Å; and (d) Xis a detectable group. The linkers may be linear or branched, maycontain aromatic moieties, and optionally may be further substituted.

Also provided is a composition including one or more molecules accordingto Formula (I) and one or more electrophoretic media.

Additionally provided herein are methods of synthesis of compounds ofthe present invention involving coupling of:

-   (a) a synthon which includes a bis-activated-ester derivative of a    detectable group; and-   (b) a synthon which includes an amine or hydrazide derivative of a    chelator; and then adding a transition metal.

Additionally provided herein are methods of synthesis of compounds ofthe present invention containing a non-sulfonated cyanine or squarainedetectable group, involving coupling of: (a) a synthon selected frommono-chelator-functionalized 2,3,3-trimethylindole,mono-chelator-functionalized 2,3,3-trimethylbenzindole,mono-chelator-functionalized 2-methyl-pyridine,mono-chelator-functionalized 2-methyl-benzothiazole,mono-chelator-functionalized 2-methyl-napthothiazole,mono-chelator-functionalized 2-methyl-benzoxazole, andmono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthon,identical or nonidentical to the synthon in (a), selected from the groupin (a); and (c) a synthon containing at least one carbon atom; and thenadding a transition metal.

Additionally provided herein are methods of synthesis of compounds ofthe present invention containing a disulfonated cyanine or squarainedetectable group, involving coupling of: (a) a synthon selected frommono-chelator-functionalized 2,3,3-trimethyl-5-sulfanato-indole,mono-chelator-functionalized 2,3,3-trimethyl-6-sulfanato-benzindole,mono-chelator-functionalized 2-methyl-5-sulfanato-pyridine,mono-chelator-functionalized 2-methyl-5-sulfanato-benzothiazole,mono-chelator-functionalized 2-methyl-6-sulfanato-napthothiazole,mono-chelator-functionalized 2-methyl-5-sulfanato-benzoxazole, andmono-chelator-functionalized 2-methyl-6-sulfanato-napthoxazole; (b) asynthon, identical or nonidentical to the synthon in (a), selected fromthe group in (a); and (c) a synthon containing at least one carbon atom;and then adding a transition metal.

Additionally provided herein are methods of synthesis of compounds ofthe present invention containing a monosulfonated cyanine or squarainedetectable group, involving coupling of: (a) a synthon selected frommono-chelator-functionalized 2,3,3-trimethylindole,mono-chelator-functionalized 2,3,3-trimethylbenzindole,mono-chelator-functionalized 2-methyl-pyridine,mono-chelator-functionalized 2-methyl-benzothiazole,mono-chelator-functionalized 2-methyl-napthothiazole,mono-chelator-functionalized 2-methyl-benzoxazole, andmono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthonselected from mono-chelator-functionalized2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized2-methyl-6-sulfanato-benzothiazole, mono-chelator-functionalized2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized2-methyl-5-sulfanato-benzoxazole, and mono-chelator-functionalized2-methyl-6-sulfanato-napthoxazole; and (c) a synthon containing at leastone carbon atom; and then adding a transition metal.

Additionally provided herein are methods of synthesis of compounds ofthe present invention containing a xanthene, xanthanone, or phenoxazinedetectable group, involving reaction of a xanthene, xanthanone, orphenoxazine detectable group, a secondary-amine derivative of achelator, and formaldehyde, according to the Mannich reaction (Mannich,C. et al. Arch. Pharm. 250:647, 1912); followed by addition of atransition metal.

Additionally provided herein is a labeled target material including atarget sequence of the form: (H)_(i), wherein H is histidine, and i is 4to 12, preferably 4 to 8, and most preferably 6, and wherein the targetsequence is bonded with a molecule according to Formula (I).

Also included is a detectable complex including a molecule according toFormula (I) and a target sequence, bonded thereto. The target sequenceincludes an amino acid sequence of the form: (H)_(i), wherein H ishistidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6.

The invention also includes a method for imparting fluorescentproperties to a target material, including the step of reacting: (a) thetarget material having a target sequence of the form (H)_(i), wherein His histidine, and i is 4 to 12, preferably 4 to 8, and most preferably6, with (b) a molecule according to Formula (I), under conditionssufficient to permit metal-chelate moieties of said molecule accordingto Formula (I) to bond to the target sequence.

Also provided is a method for detecting one or more molecules thatinclude a target sequence of the form (H)_(i), wherein H is histidineand i is an integer of from 4 to 12, the method including the steps of:(a) providing a sample including one or more molecules having a targetsequence; (b) subjecting the target material to electrophoresis in anelectrophoretic medium; (c) contacting the electrophoretic medium withat least one molecule according to Formula (I) having a detectable groupunder conditions sufficient to permit transition-metal-chelate moietiesof the molecule of Formula (I) to associate with the target sequence;and (d) detecting the detectable group, thereby detecting the one ormore molecules having a target sequence.

Furthermore, provided herein is a method for detecting a target materialof interest, including the steps of: (a) providing a target material ofinterest having a target sequence of the form: (H)_(i), wherein H ishistidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6;(b) incubating the polypeptide with a molecule according to Formula (I),having a detectable group, for a time period sufficient to allowlabeling of the target material; and (c) detecting the detectable group,thereby detecting the target material of interest.

Additionally, a method for imaging the localization, concentration orinteractions of a target material of interest on or within cells,tissues, organs or organisms is provided, including the steps of: (a)providing a target material of interest having a target sequence of theform: (H)_(i), wherein H is histidine, and i is 4 to 12, preferably 4 to8, and most preferably 6; (b) incubating the target material with amolecule according to Formula (I) for a time period sufficient to allowlabeling of the polypeptide; and (c) detecting the detectable group ofsaid molecule according to Formula (I), thereby imaging thelocalization, concentration or interactions of the target material ofinterest.

Furthermore, provided herein is an assay method for monitoring a bindingprocess including the steps of: (a) reacting a first component of aspecific binding pair with a second component of the pair, with thefirst component being labeled with a molecule according to Formula (I)having a detectable group; and (b) monitoring the reaction by monitoringa change in a signal of the detectable group.

Also provided herein is an assay method for monitoring a binding processincluding the steps of: (a) reacting a first component of a specificbinding pair with a second component of the pair, with the firstcomponent being labeled with a molecule according to Formula (I) havinga detectable group; and (b) monitoring the reaction by monitoringfluorescence emission intensity, fluorescence lifetime, fluorescencepolarization, fluorescence anisotropy, or fluorescence correlation ofthe detectable group.

Additionally provided herein is an assay method for monitoring a bindingprocess, including the steps of: (a) reacting a first component of aspecific binding pair with a second component of the pair, with thefirst component being labeled with a molecule according to Formula (I)wherein X of Formula (I) is a fluorochrome, and with the secondcomponent containing Y, wherein Y is selected from the group including afluorochrome and chromophore, Y being capable of participating influorescence energy transfer, fluorescence quenching, or excitonformation with X; and (b) monitoring the reaction by monitoringfluorescence of X.

The invention also provides an assay method for monitoring a bindingprocess, including the steps of: (a) reacting a first component of aspecific binding pair with a second component of the pair, with thefirst component being labeled with a molecule according to Formula (I)wherein X of Formula (I) is selected from the group consisting of afluorochrome and a chromophore, and with the second component containingY, wherein Y is a fluorochrome able to participate in fluorescenceenergy transfer, fluorescence quenching, or exciton formation with X;and (b) monitoring the reaction by monitoring fluorescence of Y.

The invention further provides an assay method for monitoring areaction, including the steps of: (a) reacting a first participant in areaction with a second participant in the reaction, the firstparticipant being labeled with a molecule according to Formula (I); and(b) monitoring the reaction by monitoring a change in a detectableproperty of the detectable group.

Furthermore, provided herein is a method for isolating a target materialof interest including the steps of: (a) contacting molecules accordingto Formula (I) immobilized on a solid support, with a solutioncontaining a polypeptide of interest, the polypeptide including a targetsequence of the form: (H)_(i), wherein H is histidine, and i is 4 to 12,preferably 4 to 8, and most preferably 6, under conditions that allowbinding of the target material to immobilized molecules of Formula (I);and (b) eluting the target material of interest with a low-molecularweight monothiol or low-molecular-weight dithiol.

The invention also includes a method for immobilizing a target materialof interest including the steps of: (a) contacting molecules accordingto Formula (I) immobilized on a solid support, with a solutioncontaining a target material, the target material containing a targetsequence of the form (H)_(i), wherein H is histidine, and i is 4 to 12,preferably 4 to 8, and most preferably 6, under conditions that allowbinding of the target material to immobilized molecules according toFormula (I).

Additionally, a kit for detecting a target compound is providedincluding one or more containers, wherein at least one of the containersincludes one or more molecules according to Formula (I).

Additionally provided herein is a kit including: (a) a moleculeaccording to Formula (I); and (b) a molecule containing a targetsequence including an amino acid sequence of the form: (H)_(i), whereinH is histidine, and i is an integer of from 4 to 12, preferably 4 to 8,and most preferably 6.

Further provided herein is a kit including: (a) a molecule according toFormula (I); and (b) a reagent that promotes the formation of a complexbetween the molecule according to Formula (I) and a peptide having atarget sequence of the form: (H)_(i), wherein H is histidine, and i is 4to 12, preferably 4 to 8, and most preferably 6.

Further provided is a kit including one or more containers, having oneor more molecules according to Formula (I) therein, the kit furtherincluding one or more of: (a) one or more gels; (b) one or morecontainers including molecules having a target sequence of an amino acidsequence of the form (H)_(i), wherein H is histidine and i is an integerof from 4 to 12; (c) one or more containers including antibodies havingan epitope with an amino acid sequence of the form (H)_(i), wherein H ishistidine and i is an integer of from 4 to 12; (c) one or morecontainers including one or more denaturing agents; (d) one or morecontainers including one or more buffer; and (e) one or more sets ofinstructions.

Further provided is a composition including one or more moleculesaccording to Formula (I) and one or more electrophoretic media.

Also provided is a solution for staining target molecules in anelectrophoretic medium, the solution comprising one or more moleculesaccording to Formula (I), wherein the molecules according to Formula (I)are present in a concentration sufficient to stain molecules including atarget sequence in an electrophoretic medium, the target sequenceincluding an amino acid sequence of the form (H)_(i), wherein H ishistidine and i is an integer of from 4 to 12.

Additionally, a kit including one or more containers having a stocksolution of at least one molecule of Formula (I) is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a prior-art model for the binding of neighboringhexahistidine residues to a NTA—Ni²⁺ resin.

FIGS. 2 and 3 show results of fluorescence anisotropy experimentsverifying specific interactions between bis-transition-metal-chelateprobes according to the invention with a hexahistidine-tagged protein.

FIG. 4 is a model structure of a DNA^(F)-CAP-His₆ complex showing theposition of the fluorescein of DNA^(F) (circle), the position of thehexahistidine tag of each CAP-His₆ protomotor (diamond), the distancebetween fluorescein and the hexahistidine tag of the proximal CAP-His₆protomotor (˜55 Å), and the distance between fluorescein and thehexahistidine tag of the distal CAP-His₆ protomotor (˜80 Å).

FIGS. 5 and 6 show results of FRET experiments verifying high-affinity,specific interactions of bis-transition-metal-chelate probes accordingto the present invention with a hexahistidine tagged protein.

FIGS. 7 and 8 show results of FRET experiments verifying stoichiometricinteractions of nickel containing probes according to the presentinvention with the hexahistidine tag.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventors have found, as set forth herein, that a molecule havingtwo transition-metal chelates and a detectable group binds with highaffinity and high specificity to oligohistidine target sequences,particularly hexahistidine target sequences.

Furthermore, the inventors have found that a molecule having twotransition-metal chelates and a detectable group binds with much higheraffinity (more than 10 times higher affinity) and much higherspecificity (more than 10 times higher specificity) to oligohistidinetarget sequences, particularly hexahistidine target sequences, than doesa molecule having only a single transition-metal chelate and adetectable group.

Furthermore, the inventors have found that a molecule having twotransition-metal chelates and a detectable group can be used to label,detect, and analyze target materials containing, or derivatized tocontain, oligohistidine target sequences, particularly hexahistidinetarget sequences.

Furthermore, the inventors have found that a molecule having twotransition-metal chelates and a detectable group can be used for in situlabeling, detection, and analysis of target materials containing, orderivatized to contain, oligohistidine target sequences, particularlyhexahistidine target sequences (i.e., direct labeling, detection, andanalysis of said target materials—without the need for a subsequentpurification step).

Compounds of the Invention

The present invention provides a probe for detecting a target materialof interest. The probe is a molecule including two transition-metalchelates and a detectable group, according to the following generalstructural Formula (I), and tautomers, salts, and acids thereof:

wherein: (a) Y and Y′ are each a transition metal, (b) R¹ and R^(1′) areeach independently CH(COO⁻), CH(COOH), or absent, (c) R² and R^(2′) arelinkers each having a length of about 3.0 to 20 Å, and preferably about3.0 to 15 Å, and (d) X is a detectable group. The linkers may be linearor branched, may contain aromatic moieties, and may optionally befurther substituted.

“Y” in Formula (I) is a transition metal. Y can be any transition metalcapable of specific interaction with a oligohistidine tag. Transitionmetals are those metals having incompletely filled d-orbitals andvariable oxidation states. Examples of suitable transition metalsinclude: nickel, cobalt, copper, and zinc. In a preferred embodiment, Yis a divalent transition-metal ion. In a particularly preferredembodiment, Y is selected from the group consisting of Ni²⁺, Co²⁺, Cu²⁺,and Zn²⁺.

When R¹ in Formula (I) is absent, the chelator is iminodiacetic acid(IDA). When R¹ is CH(COO—) or CH(COOH), the chelator is nitrilotriaceticacid (NTA).

Similarly, when R^(1′) in Formula (I) is absent, the chelator isiminodiacetic acid (IDA). When R^(1′) is CH(COO—) or CH(COOH), thechelator is nitrilotriacetic acid (NTA).

R² and R^(2′) in Formula (I) are linkers. The structures of R² andR^(2′) should permit the two pendant transition-metal chelates to beseparated by a distance comparable to the dimensions of a oligohistidinetarget sequence, particularly a hexahistidine target sequence. Thus, thestructures of R² and R^(2′) should permit the two pendanttransition-metal chelates to be separated by about 2.5 to 25 Å, andpreferably by about 5 to 20 Å (distances measured metal-to-metal). R²and R^(2′) may be linear or branched, may optionally contain cyclicgroups, and may optionally be further substituted. R² and R^(2′) may bethe same or different. Preferably, R² and R^(2′) are the same. R² andR^(2′) may be connected to different atoms of X (preferably two atoms onthe same edge or face of X). Alternatively, R² and R^(2′) may beconnected to the same atom of X. Alternatively, R² and R^(2′) may beconnected to a single atom, which in turn is connected, directly orthrough a linker of maximal length 4 Å, to X.

X in Formula (I) is a detectable group. “Detectable group” as usedherein refers to any chemical moiety that can be detected. Examples ofdetectable groups include fluorescent moieties, phosphorescent moieties,luminescent moieties, absorbent moieties, photosensitizers, spin labels,radioisotopes, isotopes detectable by nuclear magnetic resonance,paramagnetic atoms, heavy atoms, haptens, crosslinking agents, cleavageagents, and combinations thereof.

In one embodiment, X is detected by monitoring a signal. Some signalswhich may be monitored due to the presence of a detectable groupinclude, for example, fluorescence (fluorescence emission intensity,fluorescence lifetime, fluorescence polarization, fluorescenceanisotropy, or fluorescence correlation), luminescence, phosphorescence,absorbance, singlet-oxygen production, electron spin resonance,radioactivity, nuclear magnetic resonance, and X-ray scattering.

In another embodiment, X is detected by receptor-binding,protein-protein or protein-nucleic acid crosslinking, or protein ornucleic acid cleavage.

Preferred detectable groups include fluorescent moieties. In onepreferred embodiment, cyanine fluorescent moieties are used. Theseinclude, but are not limited to: Cy3:1-R-2-[3-[1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolium,Cy5:1-R-2-[5-[1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadienyl]-3,3-dimethyl-5-sulfo-3H-indolium, Cy7:1-R-2-[7-[1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrienyl]-3,3-dimethyl-5-sulfo-3H-indolium,indocyanine green and IRDye(1-R-2-[2-[2-R′-3-[(1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-5-sulfo-3H-indolium),and mono- and non-sulfonated derivatives thereof. In another preferredembodiment, squaraine fluorescent moieties are used. In anotherpreferred embodiment, xanthene, xanthanone, and phenoxazine fluorescentmoieties are used.

Examples of cyanine, squaraine, xanthene, xanthanone, and phenoxazinedetectable groups fluorescent moieties are described, inter alia, inSouthwick et al., 1990, Cytometry 11:418-430; Mujumdar et al., 1993,Bioconjugate Chemistry 4:105-111; Waggoner and Ernst, FluorescentRegents for Flow Cytometry, Part 1: Principles of Clinical FlowCytometry (1993) and Haugland, Molecular Probes Handbook of FluorescentProbes and Research Chemicals, Molecular Inc. 6th edition (1996) andBerling and Reiser, Methoden der Organischer Chemie, p 231-299 (1972),Oswald et al., Analytical Biochemistry 280: 272-277 (2000), Oswald etal. Photochemistry and Photobiology 74(2): 237-245 (2001), Oswald et al.Bioconjugate Chemistry 10: 925-931 (1999), U.S. Pat. No. 6,086,737. Thestructures in these publications are all incorporated herein byreference.

In a preferred embodiment, X may be selected from the following cyaninedetectable groups:

wherein U and V are each independently C(R⁴)₂, NH, O, S, or (CH)₂; R³and R^(3′) are each independently H or sulfonate; R⁴ is H, CH₃, CH₂CH₃,or (CH₂)₂CH₃; and n is 0 or an integer of from 1 to 6.

In another preferred embodiment, X may be selected from the followingsquaraine detectable groups:

wherein U and V are each independently C(R⁴)₂, NH, O, S, or (CH)₂; R³and R^(3′) are each independently H or sulfonate; R⁴ is H, CH₃, CH₂CH₃,or (CH₂)₂CH₃; R⁵ is absent or is selected from the group consisting ofH, an alkyl group, and an aryl group; and n′ is 0 or an integer of from1 to 3.

In another preferred embodiment, X may be selected from the followingxanthene, xanthanone, and phenoxazine detectable groups:

wherein R⁶, R^(6′), R^(6″), R^(6′″), R^(6″″), and R^(6′″″) are eachindependently hydrogen, halogen, hydroxyl, or alkoxyl; and R⁷, whenpresent, is hydrogen, carboxyl, carboxylate or sulfonate.

One preferred molecule of the present invention includes two pendanttransition-metal chelates and a cyanine detectable group according tothe following general structural formula:

wherein Y, Y′, R¹, R^(1′), R², and R^(2′) are as defined previously;wherein U and V are each independently C(R⁴)₂, NH, O, S, or (CH)₂; R³and R³′ are each independently H or sulfonate; R⁴ is H, CH₃, CH₂CH₃, or(CH₂)₂CH₃; and n is 0 or an integer of from 1 to 6.

Particularly preferred embodiments include the aforementioned structurewhere n is 1, 2 or 3. In an even more preferred embodiment, n is 1, 2,or 3; and R² and R^(2′) are identical and are about 3.0 to 15 Å inlength. In an especially preferred embodiment, n is 1, 2, or 3; R² andR^(2′) are identical and about 3.0 to 15 Å in length; and Y and Y′ areeach Ni²⁺.

One preferred molecule of the present invention includes two pendanttransition-metal chelates and a cyanine detectable group according tothe following general structural formula:

wherein Y and Y′ are as defined previously; U and V are eachindependently C(R⁴)₂, NH, O, S, or (CH)₂; R³ and R³⁴⁰ are eachindependently H or sulfonate; R⁴ is H, CH₃, CH₂CH₃, or (CH₂)₂CH₃; and nis 0 or an integer of from 1 to 6. In a particularly preferredembodiment, n is 1, 2, or 3; and Y and Y′ are each Ni²⁺.

Furthermore, provided herein is a molecule with two pendanttransition-metal chelates and a detectable group according to thefollowing general structural formula:

wherein Y and Y′ are as defined previously; R³ and R^(3′) are eachindependently H or sulfonate; and n is 1, 2, 3, or 4. In a particularlypreferred embodiment, n is 1, 2, or 3; and Y and Y′ are each Ni²⁺.

There are no particular limitations to the detectable groups of themolecules of the present invention, so long as the ability of thebis-transition-metal-chelate moieties to bind to a target sequence ismaintained. The point(s) of attachment between thebis-transition-metal-chelate moieties and the detectable group may vary.

Modifying groups that aid in the use of the bis-transition-metal-chelatemolecule may also be incorporated. For example, thebis-transition-metal-chelate molecule may be substituted at one or morepositions to add a solid-phase binding group or a crosslinking group.

For applications involving labeling of target materials within cells,the bis-transition-metal-chelate molecule preferably is capable oftraversing a biological membrane. Smaller molecules are generally ableto traverse a biological membrane better than larger molecules.Bis-transition-metal-chelate molecules of less than 2000 Daltons arepreferable for membrane traversal.

The polarity of the bis-transition-metal-chelate molecule can alsodetermine the ability of the bis-transition-metal-chelate molecule totraverse a biological membrane. Generally, a hydrophobicbis-transition-metal-chelate molecule is more likely to traverse abiological membrane. The presence of polar groups can reduce thelikelihood of a molecule traversing a biological membrane. Abis-transition-metal-chelate molecule that is unable to traverse abiological membrane may be further derivatized by addition of groupsthat enable or enhance the ability of the molecule to traverse abiological membrane. Preferably, such derivatization does notsignificantly alter the ability of the bis-transition-metal-chelatemolecule to react subsequently with a target sequence. Thebis-transition-metal-chelate molecule may also be derivatizedtransiently. In such instances, after traversing the membrane, thederivatizing group is eliminated to regenerate the originalbis-transition-metal-chelate molecule. Examples of derivatizationmethods that increase membrane traversability include ether formationwith acyloxyalkyl groups. For example, an acetoxymethyl ether is readilycleaved by endogenous mammalian intracellular esterases. Jansen, A. andRussell, T. J., J. Chem. Soc., 2127-2132 (1965). Also, pivaloyl ester isuseful in this regard. Madhu et al., J. Occul. Pharmaco. Ther.,14:389-399 (1998).

Methods of Synthesis of Compounds of the Invention

The invention provides methods of synthesis of compounds of the presentinvention which include coupling of: (a) a synthon which includes abis-activated-ester derivative of a detectable group; and (b) a synthonwhich includes an amine or hydrazide derivative of a chelator; and thenadding a transition metal.

The invention also provides methods of synthesis of non-sulfonatedcyanine or squaraine compounds of the present invention which includecoupling of: (a) a synthon selected from mono-chelator-functionalized2,3,3-trimethylindole, mono-chelator-functionalized2,3,3-trimethylbenzindole, mono-chelator-functionalized2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole,mono-chelator-functionalized 2-methyl-napthothiazole,mono-chelator-functionalized 2-methyl-benzoxazole, andmono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthon,identical or nonidentical to the synthon in (a), selected from the groupin (a); and (c) a synthon containing at least one carbon atom; and thenadding a transition metal.

The invention also provides methods of synthesis of disulfonated cyanineor squaraine compounds of the present invention which include couplingof: (a) a synthon selected from mono-chelator-functionalized2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized2-methyl-5-sulfanato-benzothiazole, mono-chelator-functionalized2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized2-methyl-5-sulfanato-benzoxazole, and mono-chelator-functionalized2-methyl-6-sulfanato-napthoxazole; (b) a synthon, identical ornonidentical to the synthon in (a), selected from the group in (a); and(c) a synthon containing at least one carbon atom; and then adding atransition metal.

The invention also provides methods of synthesis of monosulfonatedcyanine or squaraine compounds of the present invention which includecoupling of: (a) a synthon selected from mono-chelator-functionalized2,3,3-trimethylindole, mono-chelator-functionalized2,3,3-trimethylbenzindole, mono-chelator-functionalized2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole,mono-chelator-functionalized 2-methyl-napthothiazole,mono-chelator-functionalized 2-methyl-benzoxazole, andmono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthonselected from mono-chelator-functionalized2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized2-methyl-6-sulfanato-benzothiazole, mono-chelator-functionalized2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized2-methyl-5-sulfanato-benzoxazole, and mono-chelator-functionalized2-methyl-6-sulfanato-napthoxazole; and (c) a synthon containing at leastone carbon atom; and then adding a transition metal.

Coupling of the synthons referred to herein can be accomplished in asingle step, or in two steps. For example, for symmetric compounds(i.e., where (a) and (b) are identical), coupling of the reactants (a),(b), and (c) desirably is carried out in a single step. For asymmetriccompounds (i.e., where (a) and (b) are non-identical), coupling of thereactants (a), (b), and (c) desirably is carried out in two steps: i.e.,reaction of (a) with (c), followed by reaction of the resultant productwith (c); or, alternatively, reaction of (b) with (c), followed byreaction of the resultant product with (a).

Coupling of the synthons referred to herein can be performed insolution, or with one or more synthons attached to a solid support.

Coupling of the synthons referred to herein can be performed with thechelator in an unprotected form, or with the chelator in a protectedform initially and deprotected thereafter.

The invention also provides methods of synthesis of xanthene,xanthanone, or phenoxazine compounds of the present invention whichinclude reaction of a xanthene, xanthanone, or phenoxazine detectablegroup, a secondary-amine derivative of a chelator, and formaldehyde,according to the Mannich reaction. See, Mannich, C. et al. Arch. Pharm.250:647, (1912), the entirety of which is herein incorporated byreference. This reaction is followed by the addition of a transitionmetal.

The Mannich reaction referred to herein can be performed with thechelator in an unprotected form, or with the chelator in a protectedform initially and deprotected thereafter.

Target Materials and Target Sequences of the Invention

The invention provides detectable complexes of molecules according toFormula (I) with target sequences. Detectable complexes as used hereinrefer to the association between target amino acid sequences andbis-transition-metal-chelate molecules according to the invention.

Suitable target materials include, but are not limited to, polypeptides,and polypeptide mimetics (such as peptide nucleic acid). Preferably, thetarget material is a polypeptide.

As used herein, “polypeptide” refers to both short chains, commonlyreferred to as “peptides,” “oligopeptides,” or “oligomers,” and tolonger chains, generally referred to as “proteins”. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides may include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well-known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in research literature. Thus “polypeptide”includes peptides, oligopeptides, polypeptides and proteins, all ofwhich terms are used interchangeably herein.

The target material contains, or is modified to contain, at least onecopy of an oligohistidine target sequence, herein referred tointerchangeably as the “target sequence” or “tag”. The target sequenceis generally of the form: (H)_(i), wherein H is histidine and i is aninteger of from 4 to 12 (i.e., SEQ ID NOS. 1-9), preferably 4 to 8, andmost preferably 6.

The target sequence may be incorporated at any desired site, or set ofsites, within a target material, but preferably is incorporated at asite that is (a) accessible and (b) not essential for structure andfunction of the target material.

For example, when the target material is a protein, the target sequencepreferably is incorporated at the N-terminal region, at the C-terminalregion, at an internal loop region, at a surface-exposed non-essentialloop, at an internal linker region, or at combinations thereof. Thespecific site, or set of sites, can be chosen to accommodate thefunctional requirements of a protein. For example, it is known thatN-terminal modification of chemokines can affect their activity;therefore, in applications with chemokines, either C-terminalmodification or internal modification would be preferable. Sincelabeling is performed at defined, user-selected sites, effects on theactivity of target material can be avoided. When it is important topreserve the activity of the tagged target material, specific activitytesting of the tagged vs. the untagged target material may be conductedto verify activity. See, for example, Mas et al,. Science, 233:788-790(1986).

Target-sequence-containing polypeptides may be generated by totalsynthesis, partial synthesis, in vitro translation, or in vivobacterial, archaeal, or eukaryotic production.

In one preferred embodiment, the target sequences and/ortarget-sequence-containing polypeptides used in the invention areprepared using solid-phase synthesis (see, e.g., Merrifield et al. J.Am. Chem. Soc., 85:2149, (1962) Steward and Young, Solid Phase PeptidesSynthesis, Freeman, San Francisco, (1969), and Chan and White, FmocSolid Phase Peptide Synthesis—A Practical Approach, Oxford Press(2000)).

In another preferred embodiment, the target sequences and/ortarget-sequence-containing polypeptides used in the invention areprepared using native chemical ligation (Dawson et al., Science,266:776, 1994).

In an especially preferred embodiment, the target sequences and/ortarget-sequence-containing polypeptides are generated by in vivobacterial, archaeal, or eukaryotic expression of a recombinant nucleicacid sequence encoding the target-sequence-containing polypeptide.Methods for the construction of recombinant nucleic acid sequencesencoding a tag-containing polypeptide are well known in the art(Sambrook and Russel, Molecular Cloning A Laboratory Manual, 3^(rd) Ed.,Cold Spring Harbor Laboratory, New York (2001), the entirety of which isherein incorporated by reference). In addition, techniques for transientor stable introduction of recombinant nucleic acid sequences into cells(see, for example, Ausubel et al., Current Protocols In MolecularBiology, John Wiley & Sons, Inc. (1995)), for replacement of nativenucleic acid sequences by recombinant nucleic acid sequences in cells(see, for example, Ausubel et al., Current Protocols In MolecularBiology, John Wiley & Sons, Inc. (1995)), and for expression ofrecombinant nucleic acid sequences in cells (see e.g., Lee and Arthans,H. J. Biol. Chem., 263:3521, (1988); Rosenberg, et al., Gene, 56:125(1987)), are well known in the art.

The bis-transition-metal-chelate moieties of the molecules according toFormula (I) bind to the oligohistidine target sequence. The transitionmetals of the bis-transition-metal-chelate moieties bind to imidazolegroups of histidines of the oligohistidine target sequence.

Although not intending to be limited to such interpretation, it isbelieved that the affinity of the bis-transition-metal-chelate moleculesfor oligohistidine target sequences relates to the presence of twotridentate (where R¹ or R^(1′) is absent) or tetradentate (where R¹ orR^(1′) is CH(COO⁻) or CH(COOH)) transition-metal chelates, each having atransition metal with at least two coordination sites available forinteraction with electron-donor groups. Oligohistidine target sequencescomprising 4 to 12 histidine residues have appropriate electron-donorfunctionality, size, and flexibility to interact with availablecoordination sites of the bis-transition-metal-chelate probe, creating astable linkage therewith.

An example of a transition-metal-chelate molecule of the invention inassociation with a oligohistidine target sequence, in this case ahexahistidine target sequence, is depicted as follows:

Labeling is accomplished by contacting a bis-transition-metal-chelatemolecule according to Formula (I) with a target-sequence-containingtarget material. The bis-transition-metal-chelate molecule may becontacted with a target-sequence-containing target material located in,for example, a test tube, a microtiter-plate well, a cuvette, a flowcell, or a capillary, or immobilized on, for example a surface or othersolid support. Alternatively, the bis-transition-metal-chelate moleculemay be contacted with a target-sequence-containing target materiallocated within a cell, tissue, organ, or organism (in which embodiment,the bis-transition-metal-chelate molecule preferably is capable oftraversing an intact biological membrane).

In one embodiment, the bis-transition-metal-chelate molecules accordingto Formula (I) are used to label target-sequence-containing moleculeswithin cells. The bis-transition-metal-chelate molecules of thisinvention may be introduced into cells by diffusion (forbis-transition-metal-chelate molecules capable of traversing biologicalmembranes) or by microinjection, electroporation, or vesicle fusion (forany bis-transition-metal-chelate molecule). Thetarget-sequence-containing molecules may be introduced into cells bymicroinjection, electroporation, or vesicle fusion, or by expression ofrecombinant genes in situ.

In one preferred embodiment, a target-sequence-containing proteinproduced by expression of a recombinant gene within cells is contactedwith a molecule according to Formula (I) by incubating cells in mediumcontaining the molecule. Following labeling, and optionally followingfurther manipulations, cells are imaged using an epi-illumination,confocal, or total-internal-reflection optical microscope with anoptical detector, such as a CCD camera, an intensified CCD camera, aphotodiode, or a photomultiplier tube, and fluorescence signals areanalyzed.

Uses of the Compounds of the Invention

It is contemplated that bis-transition-metal-chelate molecules of theinvention may be used in a variety of in vitro and in vivo applications.

Desirably, the molecules of the invention will be used in separation andidentification method such as electrophoresis. Electrophoresis is apreparative and/or analytical method used to separate and characterizemacromolecules. It is based on the principle that charged particlesmigrate in an applied electrical field. If electrophoresis is carriedout in solution, molecules are separated according to their surface netcharge density. If carried out in semisolid materials (gels), however,the matrix of the gel adds a sieving effect so that particles migrateaccording to both charge and size. The particles separated in thisfashion may be stained by exposure to a sufficient concentration ofmolecules according to Formula (I) so as to render the particles,preferably biomolecules, detectable via a UV detector.

In general, electrophoresis gels can be either in a slab gel or tube gelform. For slab gels, the apparatus used to prepare them, is oftenreferred to as a cassette and usually consists of two glass or plasticplates with a space disposed between them by means of a spacer or gasketmaterial and the apparatus is held together by a clamping means so thatthe space created is closed on three sides and open at the top. Asolution of unpolymerized gel is poured into the space while in itsliquid state. A means of creating wells or depressions in the top of thegel (such as a comb) in which to place samples is then placed in thespace. The gel is then polymerized and becomes solid. Afterpolymerization is complete, the comb device is removed and the gel,while still held within the plates, is then ready for use. Examples ofsuch apparatus are well known and are described in U.S. Pat. No.4,337,131 to Vesterberg; U.S. Pat. No. 4,339,327 to Tyler; U.S. Pat. No.3,980,540 to Hoefer et al.; U.S. Pat. No. 4,142,960 to Hahn et al.; U.S.Pat. No. 4,560,459 to Hoefer; and U.S. Pat. No. 4,574,040 to Delony etal. Tube gels are produced in a similar manner, however, instead ofglass or plastic plates, glass capillary tubing is used to contain theliquid gel.

Protein electrophoresis can performed in the presence of a chargeddetergent like sodium dodecyl sulfate (SDS) which coats the surface of,and thus equalizes the surface charge of, most proteins, so thatmigration depends on size (molecular weight). Proteins are oftenseparated in this fashion, i.e., SDS-PAGE (PAGE=polyacrylamide gelelectrophoresis). One or more denaturing agents, such as urea, can alsobe included in order to minimize the effects of secondary and tertiarystructure on the electrophoretic mobility of proteins. Such additivesare typically not necessary for nucleic acids, which have a similarsurface charge irrespective of their size and whose secondary structuresare generally broken up by the heating of the gel that happens duringelectrophoresis.

Two commonly used electrophoretic media for gel electrophoresis andother separation techniques are agarose and polyacrylamide. Each ofthese is described in turn as follows. In standard PAGE technology, gelscommonly range between about 5% to about 22.5% T (T=total amount ofacrylamide or other gelling agent), mostly between about 7.5% and about15% T. Lower percentages may be employed with linear polyacrylamide. Inagarose gel electrophoresis, concentrations between about 0.2% and about2% T may be employed.

Agarose is a colloidal extract prepared from seaweed. Different speciesof seaweed are used to prepare agarose; commercially available agaroseis typically prepared from genera including, but not limited to,Gracilaria, Gelidium, and Pterocladia. It is a linear polysaccharide(average molecular mass of about 12,000) made up of the basic repeatingunit agarobiose, which comprises alternating units of galactose and3,6-anhydrogalactose. Agarose contains no charged groups and is thususeful as a medium for electrophoresis.

Agarose gels have very large “pore” size and are used primarily toseparate large molecules, e.g., those with a molecular mass greater thanabout 200 kilodaltons (kD). Agarose gels can be prepared,electrophoresed (“run”) and processed faster than polyacrylamide gels,but their resolution is generally inferior. For example, for somemacromolecules, the bands formed in agarose gels are “fuzzy” (diffuse).The concentration of agarose typically used in gel electrophoresisis isbetween from about 1% to about 3%.

Agarose gels are formed by suspending dry agarose in an aqueous, usuallybuffered, media, and boiling the mixture until a clear solution forms.This is poured into a cassette and allowed to cool to room temperatureto form a rigid gel.

Acrylamide polymers are used in a wide variety of chromatographic andelectrophoretic techniques and are used in capillary electrophoresis.Polyacrylamide is well suited for size fractionation of chargedmacromolecules such as proteins and nucleic acids (e.g.,deoxyribonucleic acids (DNA), and ribonucleic acids, (RNA)).

The creation of the polyacrylamide matrix is based upon thepolymerization of acrylamide in the presence of a crosslinker, usuallymethylenebisacrylamide (bis, or MBA). Upon the introduction of catalyst,the polymerization of acrylamide and methylene bisacrylamide proceedsvia a free-radical mechanism. The most common system of catalyticinitiation involves the production of free oxygen radicals by ammoniumpersulfate (APS) in the presence of the tertiary aliphatic amineN,N,N′,N′-tetramethylethylenediamine (TEMED). Various other chemicalpolymerization systems may be used. For example, TEMED and persulfatemay be added to provide polymerization initiation. If desired, anacrylamide gradient may be developed by successively adding solutionswith increasing amounts of acrylamide and/or cross-linking agent.Alternatively, differential initiation may be used, so as to providevarying degrees of polymerization and thus prepare a gradient gel.

Electrophoretic gels based on polyacrylamide are produced byco-polymerization of monoolefinic monomers with di- or polyolefinicmonomers. The co-polymerization with di- or polyfunctional monomersresults in cross-linking of the polymer chains and thereby the formationof the polymer network. Monoolefinic monomers include, by way ofnon-limiting example, acrylamide, methacrylamide and derivatives thereofsuch as alkyl-, or hydroxyalkyl derivates, e.g.,N-hydroxymethylacrylamide, N,N-dimethylacrylamide,N-hydroxypropylacrylamide. The di- or polyolefinic monomer is preferablya compound containing two or more acryl or methacryl groups such as e.g.methylenebisacrylamide, N,N′-diallyltartardiamide,N,N′-1,2-dihydroxyethylene-bisacrylamide, N,N-bisacrylyl cystamine,trisacryloyl-hexahydrotriazine. In a broader sense, polyacrylamide alsoincludes gels in which the monoolefinic monomer is selected fromacrylic- and methacrylic acid derivatives, e.g., alkyl esters such asethyl acrylate and hydroxyalkyl esters such as 2-hydroxyethylmethacrylate, and in which cross-linking has been brought about by meansof a compound as mentioned before. Further examples of gels based onpolyacrylamide are gels made by co-polymerization of acrylamide with apolysaccharide substituted to contain vinyl groups such as allylglycidyl dextran (see EP 0 087 995).

One type of electrophoresis is usually referred to as isoelectricfocusing (IEF) or electrofocusing. IEF, which can be carried out in anelectrophoretic medium such as a gel or a a solution, involves passing amixture through a separation medium which contains, or which may be madeto contain, a pH gradient or other pH function. The electrophoreticmedium has a relatively low pH at one end, while at the other end it hasa higher pH. IEF is discussed in various texts such as IsoelectricFocusing by P. G. Righetti and J. W. Drysdale (North Holland Publ.,Amsterdam, and American Elsevier Publ., New York, 1976).

IEF is based on the fact that the charge on a molecule, such as aprotein, depends on the pH of the ambient solution. At a pH that isequal to the isoelectric point (pI) of a specific molecule, the netcharge on that molecule is zero. At a pH above its pI, the molecule hasa negative charge, while at a pH below its pI the molecule has apositive charge. When a mixture of molecules is electrophoresed in anIEF system, an anode (positively charged) is placed at the acidic end ofthe system, and a cathode (negatively charged) is placed at the basic(alkaline) end. Each molecule having a net positive charge under theacidic conditions near the anode will be driven away from the anode. Asit moves through the IEF system, the molecule will move through zonesthat are increasingly less acid, and its positive charge will decrease.Similarly, molecules having a net positive charge under the basicconditions near the cathode will move away from the cathode, and willmove through zones that are increasingly less basic. Regardless of itsinitial starting point, each molecule will stop moving when it reachesits particular isoelectric point, since it no longer has any net chargeat that particular pH. This process thus separates molecules havingdifferent pI values. The separated molecules can be removed from the IEFmedium or from solution by various means, or they can be stained orotherwise characterized without further manipulation. For example, a gelproduced by IEF can be fixed and stained in order to detect proteinswithout removing them from the gel.

Some types of IEF systems generate pH gradients by means of syntheticampholytes (molecules having both acidic and basic characteristics) thattypically have some amount of buffering capacity. Such molecules areknown generally as “carrier ampholytes”. When placed in an IEF device,each carrier ampholyte will seek its own isoelectric point. Because oftheir buffering capacity, many carrier ampholytes will establish a pHplateau rather than a single point. By using a proper mixture of carrierampholytes, it is possible to generate a relatively smooth pH gradientfor a limited period of time. Such mixtures are sold commercially undervarious trade names, such as Ampholine™ and Pharmalyte™ (AmershamBiosciences AB, Uppsala, Sweden), SERVALYT® (SERVA Electrophoresis GmbH,Heidelberg, Germany), and Bio-Lyte (BioRad Laboratories, Hercules,Calif.). The chemistry and synthesis of ampholytes and ampholytemixtures is discussed in various references, such as U.S. Pat. No.3,485,736, U.S. Pat. No. 4,131,534, U.S. Pat. No. 5,173,160, U.S. Pat.No. 5,322,906, and U.S. Pat. No. 5,428,116; Matsui et al., Methods Mol.Biol. 112:211-219 (1999); and Lopez, Methods Mol. Biol. 112:109-110(1999).

In IEF in Immobilized pH gradients (IPG), amphoretic ions are forced toreach a steady-state position along pH inclines of various scopes andspans (see Righetti et al., Electrophoresis 15:1040-1043, 1994; Righettiet al., Methods Enzymol. 270:235-255, 1996; and 2-D Electrophoresisusing immobilized pH gradients—Principles and Methods, Edition A C,Berkelman, T. and T. Stenstedt, Amersham Biosciences, Freiburg, Germany,1998.). In one popular version of IPG, the pH gradient is in the form ofa strip and is referred to as a “gel strip” or “strip gel” that can beused in appropriate formats. See, by way of non-limiting example,published PCT patent applications WO 98/57161 A1, WO 02/09220 A1,published U.S. patent application US 2003/0015426 A1, and U.S. Pat. Nos.6,599,410; 6,156,182; 6,113,766; and 6,495,017.

Two dimensional (2D) electrophoresis techniques are also known,involving a first electrophoretic separation in a first dimension,followed by a second electrophoretic separation in a second, transversedimension. In the 2D method most commonly used, proteins are subjectedto IEF in a polyacrylamide gel in the first dimension, resulting inseparation on the basis of isolectric point, and are then subjected toSDS-PAGE in the second dimension, resulting in further separation on thebasis of size (O'Farrell, J. Biol. Chem. 250:4007-4021, 1975).

Capillary zone electrophoresis (CZE) is a technique which permits rapidand efficient separations of charged substances (for a review, seeDolnik, Electrophoresis 18:2353-2361, 1997). In general, CZE involvesintroduction of a sample into a capillary tube, i.e., a tube having aninternal diameter from about 5 to about 2000 microns, and theapplication of an electric field to the tube. The electric potential ofthe field both pulls the sample through the tube and separates it intoits constituent parts. Each constituent of the sample has its ownindividual electrophoretic mobility; those having greater mobilitytravel through the capillary tube faster than those with slowermobility. As a result, the constituents of the sample are resolved intodiscrete zones in the capillary tube during their migration through thetube. An on-line detector can be used to continuously monitor theseparation and provide data as to the various constituents based uponthe discrete zones.

CZE can be generally separated into two categories based upon thecontents of the capillary columns. In “gel” CZE, the capillary tube isfilled with a suitable gel, e.g., polyacrylamide gel. Separation of theconstituents in the sample is predicated in part by the size and chargeof the constituents traveling through the gel matrix. This technique,sometimes referred at as capillary gel electrophoresis (CGE), isreviewed by Kemp (Biotechnol. Appl. Biochem. 27:9, 1998). CGE issuitable for resolving macromolecules that differ in size but have aconstant charge-to-mass ratio (Guttman et al., Anal. Chem. 62:137,1990).

In “open” CZE, the capillary tube is filled with an electricallyconductive buffer solution. Upon ionization of the capillary, thenegatively charged capillary wall will attract a layer of positive ionsfrom the buffer. As these ions flow towards the cathode, under theinfluence of the electrical potential, the bulk solution (the buffersolution and the sample being analyzed), must also flow in thisdirection to maintain electroneutrality. This electroendosmatic flowprovides a fixed velocity component which drives both neutral speciesand ionic species, regardless of charge, towards the cathode. Fusedsilica is principally utilized as the material for the capillary tubebecause it can withstand the relatively high voltage used in CZE, andbecause the inner walls of a fused silica capillary ionize to create thenegative charge which causes the desired electroendosomatic flow. Theinner wall of the capillaries used in CZE can be either coated oruncoated. The coatings used are varied and known to those in the art.Generally, such coatings are utilized in order to reduce adsorption ofthe charged constituent species to the charged inner wall. Similarly,uncoated columns can be used. In order to prevent such adsorption, thepH of the running buffer, or the components within the buffer, aremanipulated.

The electrophoretic modalities of the invention can be carried out inany suitable format, e.g., in standard-sized gels, minigels, strips,gels designed for use with microtiter plates and other high throughput(HTS) applications, and the like. Minigel and other formats includewithout limitation those described in the following patents andpublished patent applications: U.S. Pat. No. 5,578,180, to Engelhom etal., entitled “System for pH-Neutral Longlife Electrophoresis Gel”; U.S.Pat. Nos. 5,922,185; 6,059,948; 6,096,182; 6,143,154; 6,162,338, all toUpdyke et al.; published U.S. Patent Application Nos. 20030127330 A1 and20030121784 A1; and published PCT Application WO 95/27197, all entitled“System for pH-Neutral Stable Electrophoresis Gel”; U.S. Pat. No.6,057,106, to Updyke et al., and published PCT application WO 99/37813,both entitled “Sample Buffer and Methods for High Resolution GelElectrophoresis of Denatured Nucleic Acids”; U.S. Pat. No. 6,562,213 toCabilly et al., and published PCT application WO 02/18901, both entitled“Electrophoresis Apparatus for Simultaneous Loading of MultipleSamples”; and Published U.S. Patent Application 2002/0134680 A1, toCabilly et al., and published PCT application WO 02/071024, bothentitled “Apparatus and Method for Electrophoresis”.

Any suitable buffer can be used to practice the electrophoreticmodalities of the invention. Non-limiting examples of buffers includethose described herein and in the preceding patents and published patentapplications, as well as those described in Righetti et al.,Electrophoresis 15:1040-1043 (1994); Chiari et al., Appl. Theor.Electrophor. 1:99-102 (1989); and Chiari et al., Appl. Theor.Electrophor. 1:103-107 (1989). In a particularly preferred embodiment,the buffer provided in a stock solution for use with an electrophoreticgel application will be the same as the loading buffer in the gel.

The bis-transition-metal-chelate molecules of the invention may be usedin numerous standard assay formats, as are well known in the art. Someexamples of assay formats include fluorescence emission intensity,fluorescence polarization (FP), fluorescence anisotropy (FA),fluorescence resonance energy transfer (FRET), fluorescence correlationspectroscopy (FCS), fluorescence-activated cell—or particle—sorting(FACS), x/y-fluorescence scanning (FluorImaging), epi-illuminationoptical microscopy, confocal optical microscopy,total-internal-reflection optical microscopy, absorbance spectroscopy,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),scintillation proximity assay (SPA), autoradiography, and assays formatsthat involve use of biotin or other hapten incorporation to provide arecognition event for binding or immobilization of one or morecomponents.

Some further examples, which are intended to be illustrative and notlimiting of possible assay formats and applications that could use sitespecific bis-transition-metal-chelate-labeled target materials, are setforth below.

For example, the bis-transition-metal-chelate molecules of the presentinvention may be used to detect and/or quantify a polypeptide ofinterest containing, or derivatized to contain, a target sequence. Thetarget-sequence-containing polypeptide is incubated with a moleculeaccording to Formula (I) for a time period sufficient to allow labelingthereof. Labeled target-sequence-containing polypeptide optionally maybe separated from unbound material before the detection step using anymethod known in the art, and the detectable group X is detected, therebydetecting the polypeptide of interest. The target-sequence-containingpolypeptide may be included in any material, including, but not limitedto, cuvettes, microtiter plates, capillaries, flow cells, test tubes,gels, blots, and biological samples.

The invention also provides an assay method for monitoring a bindingprocess. In this method, a first component of a specific reaction pairis labeled with a molecule according to Formula (I) and is reacted witha second component of the pair. The reaction can be monitored bymonitoring a change in a signal of the detectable group X.

Examples of specific reaction pairs include, but are not restricted to,antibodies/antigens, hormone/receptor, enzyme/substrate, andprotein/analyte.

In a fluorescence-emission-intensity assay, the sample is exposed tolight of a first wavelength (able to be absorbed by a fluorescentmoiety), and fluorescence-emission intensity is monitored at a secondwavelength (emitted by said fluorescent moiety). Fluorescence-emissionintensity is dependent on the quantity of the fluorescent moiety and onthe local environment of the fluorescent moiety.

A fluorescence-emission-intensity assay to detect and quantify bindingbetween two molecules, molecule 1 and molecule 2, may be configured asfollows: a reaction mixture is prepared by combining molecule 1 labeledwith fluorescent moiety X according to the current invention andmolecule 2. Complex formation results, directly or indirectly, from achange in the local environment of X, and, correspondingly, in a changein the fluorescence emission intensity of X. The progress of thereaction is monitored by observing the change in fluorescence emissionintensity of X. Equilibrium association and dissociation constants maybe extracted from the concentration-dependence of the reaction.

In a fluorescence-polarization (FP) or fluorescence-anisotropy (FA)assay, a sample is exposed to polarized light of a first wavelength(able to be absorbed by a fluorescent moiety), and fluorescence-emissionpolarization or anisotropy is monitored at a second wavelength (emittedby said fluorescent moiety). Fluorescence-emission polarization oranisotropy is inversely related to the rotational dynamics, and thus tothe size, of said fluorescent moiety (or, if said fluorescent moiety isattached to a molecule or complex, to the rotational dynamics, and thusto the size, of the molecule or complex). FP or FA assays permitdetection of reactions that result in changes in size of molecules orcomplexes, including especially, macromolecule-association andmacromolecule-dissociation reactions.

An FP or FA assay to detect and quantify binding between two molecules,molecule 1 and molecule 2, may be configured as follows: a reactionmixture is prepared by combining molecule 1 labeled with fluorochrome Xaccording to the current invention and molecule 2. Complex formationresults in formation of a higher-molecular-weight, higher-FP, higher-FAspecies. The progress of the reaction is monitored by observing thedecrease in FP or FA. Equilibrium association and dissociation constantsare extracted from the concentration-dependence of the reaction.

A further FP or FA assay may be used to detect and quantify proteolyticactivity and may be configured as follows: a reaction mixture isprepared by combining a substrate molecule labeled with fluorochrome Xaccording to the present invention and a sample containing a proteolyticenzyme. Cleavage of the substrate molecule by the proteolytic enzymeresults in the production of lower-molecular-weight, lower-FP, lower-FAfragments. The progress of the reaction is monitored by observing thedecrease in FP or FA.

Fluorescence resonance energy transfer (FRET) is a physical phenomenonthat permits measurement of distance). FRET occurs in a system having afluorescent probe serving as a donor and a second fluorescent probeserving as an acceptor, where the emission spectrum of the donoroverlaps the excitation spectrum of the acceptor. In such a system, uponexcitation of the donor with light of the donor excitation wavelength,energy can be transferred from the donor to the acceptor, resulting inexcitation of the acceptor and emission at the acceptor emissionwavelength. FRET readily can be detected—and the efficiency of FRETreadily can be quantified—by exciting with light of the donor excitationwavelength and monitoring emission of the donor, emission of theacceptor, or both. The efficiency of energy transfer, E, is a functionof the Förster parameter, R_(o), and of the distance between the donorand the acceptor, R:E=[1+(R/R _(o))⁶]⁻¹

wherein the Förster parameter (in angstroms, Å), is:R ₀ (in Å)=(0.211×10⁻⁵)(n ⁻⁴ Q _(DK) ² J)^(1/6)

wherein n is the refractive index of the medium, Q_(D) is the donorquantum yield in the absence of the acceptor, _(K) ² is the orientationfactor relating the donor acceptor transition dipoles, and J is thespectral overlap integral of the donor emission spectrum and theacceptor excitation spectrum.

If one performs FRET experiments under conditions where R_(o) isconstant, measured changes in E permit detection of changes in R, and,if one performs experiments under conditions where R_(o) is constant andknown, the measured absolute magnitude of E permits determination of theabsolute magnitude of R.

With fluorochromes and chromophores known in the art, FRET is usefulover distances of about 1 nm to about 15 nm, which are comparable to thedimensions of biological macromolecules and macromolecule complexes.Thus, FRET is a useful technique for investigating a variety ofbiological phenomena that produce changes in molecular proximity. WhenFRET is used as a detection mechanism, colocalization of proteins andother molecules can be imaged with spatial resolution beyond the limitsof conventional optical microscopy.

A FRET assay to detect and quantify binding between two molecules,molecule 1 and molecule 2, may be configured as follows: a reactionmixture is prepared by combining molecule 1 labeled with a moleculeaccording to Formula (I) where detectable group X is a fluorescentmoiety and molecule 2 is labeled with a fluorescent moiety Y or achrompohore Y, wherein X and Y are able to participate in FRET. Complexformation results in increased proximity between X and Y, and,correspondingly, in increased FRET. The progress of the reaction ismonitored by observing the increase in FRET. Equilibrium association anddissociation constants may be extracted from theconcentration-dependence of the reaction.

A FRET assay to detect and quantify proteolytic activity may beconfigured as follows: a reaction mixture is prepared by combining a) asubstrate molecule labeled at site 1 with Formula (I) wherein detectablegroup X is a fluorescent moiety and labeled at site 2 with fluorochromeY, wherein sites 1 and 2 are on opposite sides of theproteolytic-cleavage site, and wherein X and Y are able to participatein FRET, and b) a sample containing a proteolytic enzyme. Cleavage ofthe substrate molecule by the proteolytic enzyme results in decreasedproximity between X and Y and, correspondingly, in decreased FRET. Theprogress of the reaction is monitored by observing the decrease in FRET.

A FRET assay to detect conformation change within molecule 1 inducedupon interaction with molecule 2, may be configured as follows: areaction mixture is prepared by combining (a) molecule 1 labeled at onesite with fluorochrome X according to the current invention and labeledat another site with fluorochrome Y, wherein X and Y are able toparticipate in FRET, and (b) molecule 2. Conformation change withinmolecule 1 induced upon interaction with molecule 2 results in a changein proximity between X and Y, and, correspondingly, a change in FRET.The progress of the reaction is monitored by observing the change inFRET.

A FRET assay to measure the distance between two sites, 1 and 2, withina molecule of interest, may be configured as follows: the molecule ofinterest is labeled at site 1 with fluorochrome X according to thecurrent invention and is labeled at site 2 with fluorochrome Y, whereinX and Y are able to participate in FRET; fluorescence excitation andemission spectra are collected for X and Y; and the distance, R, iscalculated as described supra.

Fluorescence emission intensity, lifetime, polarization, aniosotropy andFRET are further described in the following references: Brand, L. andJohnson, M. L., Eds., Fluorescence Spectroscopy (Methods in Enzymology,Volume 278), Academic Press (1997), Cantor, C. R. and Schimmel, P. R.,Biophysical Chemistry Part 2, W. H. Freeman (1980) pp. 433-465. Dewey,T. G., Ed., Biophysical and Biochemical Aspects of FluorescenceSpectroscopy, Plenum Publishing (1991). Guilbault, G. G., Ed., PracticalFluorescence, Second Edition, Marcel Dekker (1990). Lakowicz, J. R.,Ed., Topics in Fluorescence Spectroscopy: Techniques (Volume 1, 1991);Principles (Volume 2, 1991); Biochemical Applications (Volume 3, 1992);Probe Design and Chemical Sensing (Volume 4, 1994); Nonlinear andTwo-Photon Induced Fluorescence (Volume 5, 1997); Protein Fluorescence(Volume 6, 2000), Plenum Publishing.

Fluorescence imaging using epi-illumination, confocal, ortotal-intemal-reflection optical microscopy permits characterization ofthe quantities, locations, and interactions of fluorochrome-labeledtarget materials within cells. All fluorescence observables that can beanalyzed in vitro—emission intensity, emission lifetime, fluorescencecorrelation, FP/FA, and FRET—also can be analyzed in cells (SeeNakanishi et al. Anal. Chem. 73:2920-2928 (2001); Maiti, S. et al. Proc.Natl. Acad. Sci. USA 94: 11753-11757 (1997); Eigen and Rigler, Proc.Natl. Acad. Sci. USA 91:5740-5747 (1994) for example of uses offluorescence in cells).

The bis-transition-metal-chelate molecules of this invention may be usedto label target-sequence-containing molecules within cells. Thebis-transition-metal-chelate molecules of this invention may beintroduced into cells by diffusion (for bis-transition-metal-chelatemolecules capable of traversing biological membranes) or bymicroinjection, electroporation, or vesicle fusion (for anybis-transition-metal-chelate molecule). The target-sequence-containingmolecules may be introduced into cells by microinjection,electroporation, or vesicle fusion, or by expression of recombinantgenes in situ.

In one embodiment, a target-sequence-containing protein produced byexpression of a recombinant gene within cells is contacted with abis-transition-metal-chelate molecule of this invention by incubatingcells in medium containing the bis-transition-metal-chelate molecule.Following labeling, and optionally following further manipulations, thecells are imaged using an epi-illumination, confocal, ortotal-intemal-reflection optical microscope with an optical detector,such as a CCD camera, an intensified CCD camera, a photodiode, or aphotomultiplier tube, and fluorescence signals are analyzed.

The fluorescent molecules of the present invention also can be used, invitro or in vivo, in single-molecule fluorescence assays withsingle-molecule detection, wherein fluorescence emission intensity,fluorescence correlation, FP/FA, or FRET is analyzed from individualsingle molecules.

The fluorescent molecules of the present invention also can be used, invitro or in vivo, in fluorescence assays with “multiplex” detection,wherein a plurality of different fluorescent molecules are attached to aplurality of different primary molecules, molecule 1a, 1b, . . . 1n,with each primary molecule being specific for a different secondarycomponent, 2a, 2b, . . . 2n, in order to monitor a plurality ofreactions between primary molecules and secondary molecules in a singlereaction mixture. According to this method of use, each of the primarymolecules is separately labeled with a fluorochrome having a different,distinguishable excitation and/or emission wavelength. The primarymolecules are then reacted, as a group, with the secondary molecules, asa group, and fluorescence is monitored at each of different,distinguishable excitation and/or emission wavelengths.

The fact that the present invention is compatible with fluorochromeshaving different, distinguishable excitation and emission wavelengths(see, e.g., Table 1 for excitation maxima and emission maxima ofderivatives of Cy3, Cy5, and Cy7 in Examples), makes the inventionparticularly important for applications involving multiplex detection.Most or all of the assays above, in vitro or in vivo, can be adapted forhigh-throughput screening, using formats, equipment, and proceduresapparent to persons skilled in the art.

Examples of fluorochromes and chromophores suitable for use in assaysabove, in conjunction with the molecules of the invention, are presentedin Haugland R. P. Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes, sixth edition (1996), ISBN 0-9652240-0-7 (Spence, MTZ,Ed). Said fluorochromes and chromophores can be incorporated intopolypeptides and other molecules of interest by any suitable method,many of which are well known in the art, including, but not limited to,chemical synthesis, enzymatic synthesis, ribosomal synthesis, chemicalligation, chemical modification, and hapten binding (see Haugland R. P.Handbook of Fluorescent Probes and Research Chemicals, supra).Alternatively, fusions of autofluorescent proteins, such as greenfluorescent protein, to a polypeptide of interest can be encoded asnucleic-acid fusion constructs, produced in cells, and analyzed in cellsor in vitro.

The methods of the invention may be used in many areas of biology andbiological research including drug screening, diagnostics and academicresearch.

It further is contemplated that the bis-transition-metal-chelatemolecules of the invention may be used for immobilization and/oraffinity-purification of target-sequence-containing molecules.

Immobilization may be accomplished by: (a) covalently attaching abis-transition-metal-chelate molecule to a surface or other solidsupport (via detectable group X or via a linker); (b) contacting theresulting bis-transition-metal-chelate-molecule-containing surface orother solid support with a solution containing atarget-sequence-containing target material; and (c) optionally washingthe surface or the solid support to remove unbound material.

Affinity purification may be accomplished by: (a) covalently attaching abis-transition-metal-chelate molecule to a surface or other solidsupport, (b) contacting the resultingbis-transition-metal-chelate-molecule-containing surface or other solidsupport with a solution containing a target-sequence-containingmolecule, (c) optionally washing the surface or other solid support toremove unbound material, and (d) eluting the target-sequence-containingmolecule with a low-molecular-weight monothiol (e.g., β-mercaptoethanol)or, preferably, a low-molecular-weight dithiol (e.g., dithiothreitol orethanedithiol).

Kits According to the Invention

In some embodiments, the molecules of the invention are prepared assolutions to be used in kits and methods such as electrophoresis.Preferably, such solutions are provided “ready-to-go,” i.e., they can beused directly to stain gels without further dilution.

In one embodiment, the molecules according to Formula (I) are providedin the form of a concentrated stock solution. The stock solution may beprovided in any convenient or suitable degree of concentration. Forexample, a stock solution may be concentrated from about 500 fold(500×), about 200×, about 100×, about 50×, about 25×, about 10×, orabout 2× more concentrated that the staining solution. That is, in orderto produce a staining solution from a 500× stock solution, the stocksolution would be diluted 500-fold. A suitable concentration may be upto about a 50 molar (M) concentration, preferably a 10M concentration,more preferably a 1M concentration. The molecules of the invention willbe provided in solution, such as in an aqueous solution. Preferably, thestock solution will include a suitable preservative, the selection ofwhich will be readily apparent to those having skill in the art. Apreferred preservative is sodium azide.

In an alternative embodiment, the molecules of the invention will beprovided in a ready-to-use form, such as a gel staining solution. Thegel staining solution is provided in a suitable concentration forimmediate use in the gel electrophoresis of interest (i.e., in aconcentration of 1×). Suitable concentrations for a gel stainingsolution are from about 0.1 μM to about 100 μM, preferably from about 1μM to about 10 μM.

In this embodiment, the molecules of the invention will be provided in asolution compatible with the particular gel electrophoresis of interest.Preferably, the gel staining solution will be supplied in solution witha buffer compatible with the particular gel electrophoresis application,more preferably the gel staining solution will be provided in a solutionhaving the same buffer as the loading buffer used in the gel.

A representative “ready-to-use” gel staining solution includes a 500 mLamber bottle containing: 500 mL of a solution of 0.2 μM of moleculesaccording to the invention in 20 mM phosphate at a pH of 7.8 and 2 mMsodium azide as a preservative.

Generally, liquid and other components of the kits are provided incontainers, which are typically resealable. The containers may betransparent, translucent or opaque. Preferably, the container for stocksolutions and gel stain solutions is an amber bottle. A preferredcontainer is an Eppendorf tube, particularly a 1.5 ml Eppendorf tube. Avariety of caps may be used with the liquid container. Generallypreferred are tubes with screw caps having an ethylene propylene O-ringfor a positive leak-proof seal. A preferred cap uniformly compresses theO-ring on the beveled seat of the tube edge. Preferably, the containersand caps may be autoclaved and used over a wide range of temperatures(e.g., +120° C. to −200° C.) including use with liquid nitrogen. Othercontainers can be used.

The invention also provides a kit including one or more moleculesaccording to Formula (I) and at least one target material including atleast one target sequence of the form: (H)_(i), wherein H is histidineand i is an integer of from 4 to 12 (i.e., SEQ ID NOS. 1-9,respectively), preferably 4 to 8, and most preferably 6.

A kit according to the invention may also generally includes at leastone molecule according to Formula (I) and at least one reagent thepromotes the formation of a complex between the molecule of Formula (I)and a target sequence of the invention.

In a further aspect, the invention relates to kits comprising one ormore molecules of the invention. Optionally, such kits further compriseone or more of the following: (a) one or more gels; (b) one or morecontainers including one or more molecules including a target sequence,the target sequence including an amino acid sequence of the form(H)_(i), wherein H is histidine and i is an integer of from 4 to 12; (c)one or more containers including one or more antibodies having anepitope having an amino acid sequence of the form (H)_(i), wherein H ishistidine and i is an integer of from 4 to 12; (d) one or morecontainers including one or more protein-denaturing solutions; (g) oneor more containers including one or more sample loading buffers; and (h)one or more sets of instructions. Optionally, the molecule of theinvention is provided on one or more solid supports. The solid supportsare preferably a purification column, a cellulose blot or a bead.Optionally, one or more of the additional liquid elements of the kit areprovided in an appropriate container such as a sealed vial or the like.

Antibodies used in the kits will have an epitope include an amino acidsequence of the form (H)_(i), wherein H is histidine and i is an integerof from 4 to 12 include. Examples of suitable epitopes, by way ofnon-limiting example, include polyclonal antibodies, such as thoseavailable from Medical and Biological Laboratories, Inc. (Nagoya,Japan); monoclonal antibodies (mAbs) such as 4D11 (Abcam, Inc.;Cambridge, Mass.), 27E8 (Cell Signaling Technology, Inc.; Beverly,Mass.), IPA2C6.1 (Exalpha Biologicals, Inc.; Watertown, Mass.), andthose described in published PCT application WO 96/26963 (EMDBiosciences, Inc., Novagen Brand, Madison, Wis.); other mAbs availablefrom, e.g., Oncogene Research Products (San Diego, Calif.), BDBiosciences Clontech (Palo Alto, Calif.), and others; and single-chainantibodies, such as that derived from the anti-His tag antibody 3D5(Kaufmann et al., J Mol Biol. 318:135-147, 2002).

One type of kit of the invention comprises a solution comprising one ormore molecules according to Formula (I), wherein the one or moremolecules are present in a concentration sufficient to stain moleculesincluding a target sequence, the target sequence comprising an aminoacid sequence of the form (H)_(i), wherein H is histidine and i is aninteger of from 4 to 12, and wherein said target molecules are in anelectrophoretic medium.

A typical method for staining using electrophoretic media in a gelformat that can be carried out at ambient temperature includes the stepsof: fixing the gel (e.g., incubating the gel in an aqueous solutionhaving 40% ethanol and 10% acetic acid for about 1 hour); rinsing thefixed gel one or more times with distilled water for about 10 minutes;incubating the gel in a staining solution for about 1 hour; and washingthe gel one or more times with 20 mM sodium phosphate, pH 7.8. Forvisualization, the gel is placed, for example, on a transilluminatoroperating at a wavelength of 302 nm uv.

Typically, the fixing solution contains a polar organic solvent, forexample, an alcohol. Preferably, the polar organic solvent is an alcoholhaving 1-6 carbon atoms, or a diol or triol having 2-6 carbon atoms.Preferred alcohols are methanol or ethanol mixed with acetic acid. Thealcohols, in one illustrative example, are present in an aqueoussolution of about 50% ethanol or methanol with 10% acetic acid. The gelcan be fixed in multiple sequential steps. Essentially, the gel isimmersed in the fixing solution for example, for at least 20 minutes andthen removed from the solution and new solution added for at least 3hours and up to 24 hours.

The staining solution can be prepared in a variety of ways, which isdependent on the medium the sample is in. A particularly preferredstaining solution is one that is formulated for detection of affinitytags in a gel. Specifically, the staining solution comprises afluorescent compound of the present invention in an aqueous solution;optionally the staining solution comprises an organic solvent and abuffering component. The selection of the fluorescent compound dictates,in part, the other components of the staining solution. Any of thecomponents of the staining solution can be added together or separatelyand in no particular order wherein the resulting staining solution isadded to the gel. Alternatively, the components of the staining solutioncan be added to a gel in a step-wise fashion. The fluorescent compoundis prepared by dissolving it in a solvent, such as water, DMSO, DMF ormethanol. Usually the fluorescent compound is at a final concentrationof about 0.01 μM to 100 mM, for example, 0.05 μM to 10 μM, in certainillustrative examples, from 0.05 μM to 1 μM. In another example, thecompound is present at a final concentration of 0.1 μM to 0.3 μM, morespecifically 0.2 μM.

In certain illustrative embodiments, the pH of the staining solution is7-10, more specifically, for example, the pH is 7.1 to 10.0, 7.4 to 8.8,7.6 to 8.2, 7.7 to 7.9, or 7.8. The pH of the staining mixture isoptionally modified by the inclusion of a buffering agent in addition toor in place of an alkaline component. Any buffering agent that maintainsan alkaline environment and is compatible with the affinity tag andfusion protein in the sample is suitable for inclusion in the stainingmixture. For example, buffers used in the staining mixture can beTricine, BES, phosphate or Tris. In certain illustrative embodiments,the buffer is phosphate or Tris. In addition, the staining solution caninclude an effective amount of a preservative. Many preservatives areknown in the art and can be used with the present invention. Forexample, sodium azide can be included in the staining solution.

Optionally, the staining solution contains a metal ion salt. Nickel ionsand cobalt ions have affinity for both the bis-transition metal chelateprobes of the present invention and the poly-histidine affinity tag,therefore nickel or cobalt salts are optionally included in stainingsolutions of the present invention. An exemplified salt is nickelchloride but any nickel or cobalt salt known to one skilled in the artcan be used. The salt is typically present in the staining solution at aconcentration of about 10 nm to 1 mM; preferably the concentration isabout 1 μM to 200 μM.

After staining and before detection (i.e. visualization), the gel can berinsed in a rinse solution that is similar in composition, or identicalin composition, to the staining solution except that it does not includethe bis-transition-metal-chelate compound of the present invention.

EXAMPLE 1

Synthesis of (Ni²⁺-NTA)₂-Cy3

A. Synthesis of (NTA)₂-Cy3

N-(5-amino-1-carboxypentyl)iminodiacetic acid (Dojindo; 26 mg, 80 μmol)was dissolved in 1.6 ml 0.1M sodium carbonate and was added to Cy3bis-succinimidyl-ester (“Cy3 Reactive Dye” from Amersham-PharmaciaBiotech). Following reaction for 1 hour (with vortexing at 15-minintervals) at 25° C. in the dark, products were purified from excessN-(5-amino-1-carboxypentyl)iminodiacetic acid using a Sep-Pak C18cartridge ((Millipore; pre-washed with 10 ml of acetonitrile and 10 mlwater; washed with 20 ml water; eluted with 1 ml 60% methanol), dried,re-dissolved in 200 μl methanol, and purified by preparative TLC [1000 Åsilica gel (Analtech); NH₄OH:ethanol:water 55:35:10 v/v/v]. Three bandswere resolved, corresponding to (NTA)₂-Cy3 (r_(f)=0.2), (NTA)₁-Cy3 monoacid (r_(f)=0.5), and (NTA)₂-Cy3 bis acid (r_(f)=0.8). (NTA)₂-Cy3 waseluted using 60% methanol, dried, re-dissolved in 2 ml water andquantified spectrophotometrically (ε₅₅₀-150,000M⁻¹ cm⁻¹). The yield was64 nmol, 8%. ES-MS: m/e 1197.0 (calculated 1197.4).

B. Synthesis of (Ni²⁺-NTA)₂-Cy3

NiCl₂ (Aldrich; 350 μmol of NiCl₂ in 3 μl of 0.01N HCl) was added to(NTA)₂-Cy3 (70 nmol in 2 ml water), and the solution was brought to pH 7by addition of 0.8 ml 50 mM sodium acetate (pH 7), 200 mM NaCl.Following reaction for 30 min. at 25° C. in the dark, the product waspurified using a Sep-Pak C18 cartridge ((Millipore; procedure as above)and dried. ES-MS: m/e 1316.8 (calculated 1315.7). Ni²⁺ content[determined by performing analogous reaction with ⁶³NiCl₂ (New EnglandNuclear) and quantifying reactivity in product by scintillation countingin Scintiverse II (Fischer)]: 1.4 mol Ni²⁺ per mol. Spectroscopicproperties are reported in Table 1. TABLE 1 (XXV)

Spectroscopic Properties of (Ni²⁺—NTA)₂—Cy3 and Ni²⁺—NTA)₂—Cy3^(a)fluorochrome λ_(max, exc)(nm) λ_(max, em)(nm) quantum yield (Q)(Ni²⁺—NTA)₂—Cy3 552 565 0.04 (Ni²⁺—NTA)₂—Cy5 650 668 0.05^(a)Ni²⁺-free analogues exhibit identical λ_(max, exc) and λ_(max, em)and 3.8-fold higher Q (with the higher Q presumably reflecting theunavailability of nonradiative decay involving Ni²⁺ unoccupied dorbitals).

EXAMPLE 2

Synthesis of (Ni²⁺-NTA)₂-Cy5

A. Synthesis of (NTA)₂-Cy5

N-(5-amino-1-carboxypentyl)iminodiacetic acid (Dojindo; 40 mg; 125 μmol)was dissolved in 0.8 ml 0.1M sodium carbonate and was added to Cy5bis-succinimidyl-ester (“Cy5 Reactive Dye” Amersham-Pharmacia Biotech;800 nmol). Following reaction for 1 h (virtexed at 15 minute intervals)at 25° C. in the dark, products were purified from excessN-(5-amino-1-carboxypentyl)iminodiacetic acid using a Sep-Pak C18cartridge (Millipore; procedure as above), dried, re-dissolved in 200 μlmethanol, and purified in 100 μm portions by preparative TLC [silicagel, 1000 Å (Analtech); NH₄OH:ethanol:water in a 55:35:10 v/v/v. Threebands were resolved, corresponding to (NTA)₂-Cy5 (r_(f)=0.2), (NTA)₁-Cy5mono acid (r_(f)=0.6), and (NTA)₂-Cy5 bis acid (r_(f)=0.8). The(NTA)₂-Cy5 was eluted with 60% methanol, dried, re-dissolved in 2 mlwater and quantified spectrophotometrically (ε₅₅₀=250,000M⁻¹ cm⁻¹).Yield: 60 nmol; 7.5%.

B. Synthesis of (Ni²⁺-NTA)₂-Cy5

NiCl₂ (Aldrich; 90 nmol in 1 μl of 0.01 N HCl) was added to (NTA)₂-Cy5(30 mmol in 1 ml water), and the solution was bought to pH 7 by additionof 0.5 ml 50 mM sodium acetate (pH 7), 70 mM NaCl. Following reactionfor 30 min. at 25° C. in the dark, the product was purified using aSep-Pak C18 cartridge (Millipore; procedure as above) and dried. ES-MS:mle 1341.0 (calculated 1341.7). Spectroscopic properties are reported inTable 1.

EXAMPLE 3

Preparation of a C-Terminally Hexahistidine Tagged Derivative of theTranscriptional Activator CAP (CAP-His₆)

A. Preparation of CAPHis₆

Plasmid pAKCRP-His₆ encodes CAP-His₆ under the control of bacteriophageT7 gene 10 promotor. Plasmid AKCRP-His₆ was constructed from plasmidpAKCRP (as described in Kapanidis, A. et al., J. Mol. Biol. 312:453-468(2001) by using site-directed mutagenesis (as described in Kukel, etal., J. Meths. Enzymol., 204:125-138 (1991)) to insert six His codons(CAC-CAC-CAC-CAC-CAC-CAC) after codon 209 of the crp gene.

To prepare CAP-His₆, a culture of E. coli strain BL21(DE3) (Novagen)transformed with pAKCRP-His₆ was shaken at 37° C. in 1 L LB (asdescribed in Miller, J., Experiments in Molecular Genetics, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1972)) containing 200 mg/mlampicillin until OD₆₀₀=0.5, induced by addition ofisopropyl-thio-β-D-galactoside to 1 mM, and shaken an additional 3 h at37° C. The culture was harvested by centrifugation (4,500×g; 15 min. at4° C.), the cell pellet was re-suspended in 15 ml buffer A [20 mMTris-HCl (pH 7.9), 500 mM NaCl, 5 mM imidazole], cells were lysed bysonication, and the lysate was cleared by centrifugation (30,000×g; 30min. at 4° C.). The sample was adjusted to 15 ml with buffer A, adsorbedonto 2 ml Ni²⁺-NTA agarose (Qiagen) in buffer A, washed with 12 mlbuffer A containing 20 mM imidazole, and eluted with 6×1 ml buffer Acontaining 200 mM imidazole.

Fractions containing CAP-His₆ were pooled, desalted twice into buffer B[40 mM Tris-HCl (pH 8), 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol]by gel-filtration chromatography on NAP-10 (Amersham-Pharmacia Biotech),quantified spectrophotometrically (ε_(278, protomer)=20,000 M⁻¹ cm⁻¹),and stored in aliquots at −80° C. Yield ˜20 mg/L culture. Purity >99%.

EXAMPLE 4

Verification of Affinity and Specificity of Association of(Ni²⁺-NTA)₂Cy3 and (Ni²⁺-NTA)₂Cy5 with Target Material

Affinity and specificity of association of the probe with targetmaterial were evaluated using fluorescence anisotropy assays (methods asin Jameson and Dwyer, Methods Enzymol., 246:283-300 (1995)). Formationof a complex of the probe with a His₆-tagged protein was detected as anincrease in fluorescence anisotropy, A, arising from the increase inmolecular size and corresponding decrease in rotational dynamics.

Reaction mixtures [200 μl, in 100 μl quartz micro-cuvettes (Starna)]contained 50 nM of (Ni²⁺-NTA)₂-Cy3 or (Ni²⁺-NTA)₂-Cy5 in buffer C [40 mMTris-HCl (pH 8), 100 mM NaCl, 1 mM dithiothreitol, 0.5 mM imidazole, 0.2mM cAMP, 100 μg/ml bovine serum albumin, and 5% glycerol]. Reactionmixtures were titrated with 0-3 μM CAP-His₆ (or CAP) by successiveaddition of 0.5-4 μl aliquots of 2-4 μM CAP-His₆ (or CAP) in the samebuffer. Fluorescence anisotropy was determined at the start of thetitration and 5 min after each successive addition in the titration. Allsolutions were maintained at 25° C.

Fluorescence measurements were performed using a commercial steady-statefluorescence instrument (QM-1, PTI) equipped with T-format Glan-Thompsonpolarizers (PTI). Excitation wavelengths were 530 nm for (Ni²⁺-NTA)₂-Cy3and 630 nm for (Ni²⁺-NTA)₂-Cy5; emission wavelengths were 570 nm for(Ni²⁺-NTA)₂-Cy3 and 670 nm for (Ni²⁺-NTA)₂-Cy5. Slit widths were 10 nm.Fluorescence emission intensities were corrected for background bysubtraction of fluorescence emissions intensities for control reactionscontaining identical concentrations of CAP-His₆ or CAP but notcontaining probe.

Fluorescence anisotropy, A, was calculated using:A=(I_(VV)−GI_(VH))/(IVV+2G_(VH)) where I_(VV) and I_(VH) are thefluorescent intensities with the excitation polarizer at a verticalposition and the emission polarizers at vertical and horizontalpositions, respectively, and G is the grating correction factor. Datawere plotted as: (A−A₀/A₀) where A is the fluorescence anisotropy in thepresence of the indicated concentration of CAP-His₆ or CAP, and A₀ isthe fluorescence anisotropy in the absence of CAP-His₆ or CAP.Equilibrium dissociation constants were calculated using linearregression.

Referring now to FIG. 2, a graphical representation of results oftitration of (Ni²⁺-NTA)₂-Cy3 with His₆-CAP is shown (filled circles).Specific interaction between (Ni²⁺-NTA)₂-Cy3 and CAP-His₆ is evidencedby a large, saturable increase in fluorescence anisotropy. High affinityof interaction is evidenced by a low equilibrium dissociation constant(K_(D)=1.0 μM). Specificity of interaction is evidenced by the absenceof a significant increase in fluorescence anisotropy in a controltitration with CAP (open circles; >95% specificity).

Referring now to FIG. 3, a graphical representation is shown oftitration of (NTA)₂-Cy5 with CAP-His₆ is shown (filled circles).Specific interaction between (Ni²⁺-NTA)₂-Cy5 and His₆-CAP is evidencedby a large, saturable increase in fluorescence anisotropy. High affinityof interaction is evidenced by a low equilibrium dissociation constant(K_(D)=0.4 μM). Specificity of interaction is evidenced by the absenceof a significant increase in fluorescence anisotropy in a controltitration with CAP (open circles; (>95% specificity).

EXAMPLE 5

Verification of Affinity, Specificity, and Stoichiometry of Associationof (Ni²⁺-NTA)₂-Cy3 and (Ni²⁺-NTA)₂-Cy5 with Target Material: FRET

The affinity, specificity, and stoichiometry of interactions betweenprobes according to the invention and His₆ also were verified using FRETassays. A His₆-tagged protein-DNA complex, (CAP-His₆)-DNA^(F), wasprepared. FRET assays using the probes according to the invention thenwere performed to verify interactions, to detect a target material, andto measure an intermolecular distance.

A. Preparation of DNA^(F)

DNA^(F), 53 base pair fluorescein-labelled DNA fragment containing theconsensus DNA site for CAP (fluorescein incorporated at position -9relative to the consensus DNA site for CAP) was prepared as described inEbright, R. et al., J. Mol. Biol. 312:453-468 (2001).

B. FRET Assays-Standard Titrations

Reaction mixtures (200 μl, in 50 μl quartz micro-cuvettes (Starna))contained 5 nM DNA^(F) and 50 nM CAP-His₆ (or CAP) in buffer C. Reactionmixtures were titrated with 0-3.2 μM of (Ni²⁺-NTA)₂-Cy3 or(Ni²⁺-NTA)₂-Cy5 by successive addition of 0.3-1.2 μl aliquots of 30-300μM of (Ni²⁺-NTA)2-Cy3 or (Ni²⁺-NTA)₂-Cy5 in the same buffer.Fluorescence emission was determined at the start of the titration and 5min after each successive addition in the titration. All solutions weremaintained at 25° C.

Fluorescence emission intensities, F, were measured using a commercialsteady-state fluorescence instrument (QM-1, PTI) equipped with T-formatGlan-Thompson polarizers (PTI) set at 54.7° (“magic angle”). Excitationwavelength was 480 nm; emission wavelength range was 500-600 nm(titrations with (Ni²⁺-NTA)₂-Cy3) or 500-700 nm (titrations with(Ni²⁺-NTA)₂-Cy5; excitation slit width was 10 nm; emission slit widthwas 15 nm. Fluorescence emission intensities were corrected forbackground (by subtraction of fluorescence emission intensities forcontrol reaction mixtures containing identical concentrations of(Ni²⁺-NTA)₂-Cy3 or (Ni²⁺-NTA)₂-Cy5, but not containing CAP-His₆ or CAP)and for dilution.

Efficiencies of FRET, E, were calculated as:E=1−(F^(520,480)/F^(520/480) _(o)) where F^(520,480) is the fluorescenceemission intensity of the fluorescein label at the indicatedconcentration of (Ni²⁺-NTA)₂-Cy3 or (Ni²⁺-NTA)₂-Cy5 and F^(520/480) _(o)is the fluorescence emission intensity of the fluorescein label at 0 μMof (Ni²⁺-NTA)₂-Cy3 or (Ni²⁺-NTA)₂-Cy5. Data were plotted as E vs.titrant concentration, and binding curves and equilibrium dissociationconstants were calculated using non-linear regression (as described inGunasekera, A. et al., J. Biol. Chem., 267:14,713-14,720 (1992)).

Referring now to FIG. 5, a graphical representation of results oftitration of the (CAP-His₆)-DNA^(F) complex with (Ni²⁺-NTA)₂-Cy3 isshown (filled circles). Specific interaction between the(CAP-His₆)-DNA^(F) complex and (Ni²⁺-NTA)₂-Cy3 is evidenced by a large,saturable increase in FRET. High affinity of interaction is evidenced bya low equilibrium dissociation constant (K_(D)=0.9 μM). Specificity ofinteraction is evidenced by the absence of a significant increase inFRET in a control titration with the CAP-DNA^(F) complex (open circles;(>95% specificity).

Referring now to FIG. 6, a graphical representation of results oftitration of the (CAP-His₆)-DNA^(F) complex with (Ni²⁺-NTA)₂-Cy5 isshown (filled triangles). Specific interaction between the(CAP-His₆)-DNA^(F) complex and (Ni²⁺-NTA)₂-Cy5 is evidenced by a large,saturable increase in FRET. High affinity of interaction is evidenced bya low equilibrium dissociation constant (K_(D)=0.3 μM). Specificity ofinteraction is evidenced by the absence of a significant increase inFRET in a control titration with the CAP-DNA^(F) complex (opentriangles; >95% specificity).

C. FRET Assays-Stoichiometric Titrations

Stoichiometric titrations were performed analogously to standardtitrations (as described in Example 5B), using reaction mixturescontaining 0.6-2.6 μM (CAP-His₆)-DNA^(F) [prepared by equilibration ofDNA^(F) with excess CAP-His₆ for 20 min. at 25° C., followed by removalof unbound CAP-His₆ by filtration through Bio-Rex 70 (Bio-Rad),accordingto methods described in Kapanidis, A. N., et al., J. Mol. Biol.312:453-468 (2001)], and titrating with 0-12 μM of (Ni²⁺-NTA)₂-Cy3 or(Ni²⁺-NTA)₂-Cy5 by successive addition of 0.3-1.2 μl aliquots of μM(Ni²⁺-NTA)₂-Cy3 or (Ni²⁺-NTA)₂-Cy5. Fluorescence emission intensitieswere corrected for dilution and background, and values of E werecorrected for non-specific interactions (by subtraction of values of Efor control reaction mixtures omitting CAP-His₆). Corrected values of Ewere plotted as E/E_(sat) vs. titrant concentration where E_(sat) is theE at saturating titrant concentrations.

Referring now to FIG. 7, a graphical representation of results ofstoichiometric titration of the (CAP-His₆)-DNA^(F) complex with(Ni²⁺-NTA)₂-Cy5 is shown (filled circles). The interaction between with(Ni²⁺-NTA)₂-Cy5 and His₆ has a stoichiometry is 1:1, as evidencedinflection of the titration curve at a ratio of 1 mole (Ni²⁺-NTA)₂-Cy5to 1 mole CAP-His₆ protomer.

Referring now to FIG. 8, a graphical representation of results ofstoichiometric titration of the (CAP-His₆)-DNA^(F) complex with(Ni²⁺-NTA)₂-Cy3 is shown (filled circles). The interaction between with(Ni²⁺-NTA)₂-Cy3 and His₆ has a stoichiometry is 1:1, as evidencedinflection of the titration curve at a ratio of 1 mole (Ni²⁺-NTA)₂-Cy3to 1 mole CAP-His₆ protomer.

D. FRET Assays-Distance Determinations

Donor-acceptor distances, R, were determined using the measuredefficiencies of FRET at saturation, E_(sat) (0.45 for titration with(Ni²⁺-NTA)₂-Cy5; 0.25 for titration (Ni²⁺-NTA)₂-Cy5; see FIGS. 5, 6),and the measured Förster parameters, R₀:E=R ₀ ⁶/(R ₀ ⁶ +R ⁶)R ₀ (in Å)=(0.2 11×10⁻⁵)(n ⁻⁴ Q _(DK) ² J)^(1/6)where n is the refractive index of the medium (1.4 for dilute proteinsolutions⁸), Q_(D) is the donor quantum yield in the absence of acceptor[0.4; measured using quinine sulfate in 0.1 N N₂SO₄ as standard(Q_(QS)=0.51)], _(K) ² is the orientation factor relating the donoremission dipole and acceptor dipole [approximated as ⅔ due to the lowfluorescent anisotropy of the donor], and J is the spectral overlapintegral of the donor emission spectrum and the acceptor excitationspectrum:J=[∫F _(D)(λ)ε_(A)(λ)λ⁴ dλ]/[∫F _(D)(λ)dλ]where F_(D)(λ) is the normalized corrected emission spectrum of donor,ε_(A)(λ) is the molar extinction coefficient of acceptor, and λ is thewavelength.

The analysis above yields donor-acceptor distances of 58 Å using(Ni²⁺-NTA)₂-Cy3; FIG. 5) and 53 Å (using (Ni²⁺-NTA)₂-Cy5; FIG. 6). Thedistance of 56(±) Å determined in this manner is in excellent agreementwith the distance of about 55 Å expected based on structural informationas illustrated in FIG. 4 (corresponding to the distance between thefluorescein on DNA and the His₆ of the proximal CAP-His₆ protomer).

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitations,which is not specifically disclosed herein. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed herein, optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. In addition, where features or aspects of the inventionare described in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other aspects ofthe invention are within the following claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1-81. (canceled).
 82. A method for detecting one or more molecules that include a target sequence, wherein said target sequence comprises an amino acid sequence of the form (H)_(i), wherein H is histidine and i is an integer of from 4 to 12, said method comprising: (a) providing a sample that comprises one or more target material, wherein said target material includes molecules having a target sequence, (b) subjecting said target material to electrophoresis in an electrophoretic medium; (c) contacting said electrophoretic medium with at least one molecule according to Formula (I) having a detectable group under conditions sufficient to permit transition-metal-chelate moieties of said molecule of Formula (I) to associate with said target sequence; and (d) detecting said detectable group, thereby detecting said one or more molecules that include a target sequence.
 83. The method of claim 82, wherein said electrophoresis is selected from the group consisting of solution electrophoresis, SDS-PAGE, IEF, IPG electrophoresis, and 2D electrophoresis. 84-86. (canceled).
 87. A solution for staining target molecules in an electrophoretic medium, said solution comprising one or more molecules according to Formula (I), wherein said one or more molecules are present in a concentration sufficient to stain molecules including a target sequence in an electrophoretic medium, said target sequence comprising an amino acid sequence of the form (H)_(i), wherein H is histidine and i is an integer of from 4 to
 12. 88-89. (canceled).
 90. The method of claim 82, wherein the electrophoretic medium is contacted with the at least one molecule according to Formula (I) in a solution having a pH of between 7.1 and 8.0.
 91. The method of claim 82, wherein the electrophoretic medium is contacted with the at least one molecule according to Formula (I) in a solution having a pH of between 7.6 and 8.2.
 92. The method of claim 82, wherein the electrophoretic medium is contacted with the at least one molecule according to Formula (I) in a solution having a pH of between 7.6 and 8.0.
 93. The method of claim 90, wherein the at least one molecule according to Formula (I) is present at a concentration from 0.05 μM to 1 μM.
 94. The method of claim 90, further comprising a preservative.
 95. The method of claim 90, further comprising a phosphate buffer or a Tris buffer.
 96. The method of claim 82, wherein the electrophoretic medium is contacted with the at least one molecule according to Formula (I) in a solution comprising a phosphate buffer.
 97. The method of claim 90, further comprising a metal ion salt.
 98. The solution of claim 87, wherein the solution has a pH of between 7.1 and 8.0.
 99. The solution of claim 87, wherein the solution has a pH of between 7.6 and 8.2.
 100. The solution of claim 87, wherein the solution has a pH of between 7.6 and 8.0.
 101. The solution of claim 98, wherein the at least one molecule according to Formula (I) is present at a concentration from 0.05 μM to 1 μM.
 102. The solution of claim 98, further comprising a preservative.
 103. The solution of claim 98, further comprising a phosphate buffer or a Tris buffer.
 104. The solution of claim 87, wherein the solution comprises a phosphate buffer.
 105. The solution of claim 98, further comprising a metal ion salt.
 106. The solution of claim 98, wherein the solution is present in a ready-to-use form in a container. 